Method for producing purine nucleosides and nucleotides by fermentation using a bacterium belonging to the genus Escherichia or Bacillus

Abstract

A method is provided for producing a purine nucleoside, such as inosine and guanosine, and a method for producing a 5′-purine nucleotide such as 5′-inosinic acid or 5′-guanylic acid, using a bacterium belonging to the either genus Escherichia or genus Bacillus , wherein purine nucleoside productivity of said bacterium is enhanced by enhancing an activity of a protein encoded by the yeaS (leuE) gene.

1. A method for producing a purine nucleoside comprising cultivating a bacterium of the genus  Escherichia  or  Bacillus  having a purine nucleoside producing ability in a culture medium, and collecting from the culture medium the purine nucleoside, wherein an activity of YeaS protein is enhanced in the bacterium, the YeaS protein is selected from the group consisting of:
 A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2; and 
 B) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, wherein one to five amino acids are substituted, deleted, inserted, added, or inverted, and the protein has the activity of YeaS protein, wherein the activity is making the  Escherichia  or  Bacillus  bacterium resistant to 8-azaadenine, 2,6-diaminopurine, 6-mercaptopurine, or 6-thioguanine. 
 
     
     
       2. The method according to  claim 1 , wherein the bacterium has been modified to have enhanced expression of a gene involved in purine nucleoside biosynthesis. 
     
     
       3. The method according to  claim 1 , wherein the purine nucleoside is inosine or guanosine. 
     
     
       4. The method according to  claim 1 , wherein the activity of the protein is enhanced by transforming the bacterium with a DNA coding for the protein, or by modifying an expression control sequence of a DNA coding for the protein on the chromosome of the bacterium so that the expression of the DNA coding for the protein is enhanced, wherein said DNA comprises the nucleotide sequence of SEQ ID NO: 1. 
     
     
       5. The method according to  claim 4 , wherein said DNA is located on a multicopy vector. 
     
     
       6. A method for producing a 5′-purine nucleotide comprising cultivating a bacterium of the genus  Escherichia  or  Bacillus  having a purine nucleoside producing ability in a culture medium to produce a purine nucleoside, phosphorylating the purine nucleoside to generate a purine nucleotide, and collecting the 5′-purine nucleotide, wherein an activity of YeaS protein is enhanced in the bacterium, the YeaS protein is selected from the group consisting of:
 A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2; and 
 B) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, wherein one to five amino acids are substituted, deleted, inserted, added, or inverted, and the protein has the activity of YeaS protein, wherein the activity is the activity of making the  Escherichia  or  Bacillus  bacterium resistant to 8-azaadenine, 2,6-diaminopurine, 6-mercaptopurine, or 6-thioguanine. 
 
     
     
       7. The method according to  claim 6 , wherein the bacterium has been modified to have enhanced expression of a gene involved in purine nucleoside biosynthesis. 
     
     
       8. The method according to  claim 6 , wherein the purine nucleoside is inosine and/or guanosine, and the 5′-nucleotide is inosinic acid and/or guanylic acid. 
     
     
       9. The method according to  claim 6 , wherein the activity of the protein is enhanced by transforming the bacterium with a DNA coding for the protein, or by modifying an expression control sequence of a DNA coding for the protein on the chromosome of the bacterium so that the expression of the DNA coding for the protein is enhanced, wherein said DNA:
 comprises the nucleotide sequence of SEQ ID NO: 1. 
 
     
     
       10. The method according to  claim 9 , wherein said DNA is located on a multicopy vector.